Methods to detect and quantify RNA

Abstract

Improved methods to quantitate RNA in biological or other analytical samples employ extended RNAs containing adaptors at the 5′ end and polyA sequences coupled to a tag at the 3′ end. The invention method is particularly useful in quantitating microRNAs as primers can be used that need not complement the non-conserved 3′ ends of these molecules.

The invention claimed is: 
     
       1. A method to determine the quantity of a target RNA in a sample, which method comprises:
 providing extended molecules of said target RNA in said sample, wherein said molecules have been extended at the 5′ terminus by a 5′ adaptor nucleotide sequence and extended at the 3′ terminus by poly A; 
 converting said extended target RNA into cDNA by reverse transcription using as primer an oligomer with the sequence: 3′-two-base-anchor-polyT-tag-5′ to obtain an RNA:cDNA duplex; 
 directly annealing said RNA:cDNA duplex with a first primer consisting of 3′-sequence complementary to sequence at the 3′ end of the target RNA, deoxynucleotide sequence complementary to polyA at the 3′ terminus of the extended target RNA and optionally sequence of said tag and with a second primer that consists of 5′ deoxynucleotide sequence identical to the sequence of the 5′ adaptor and 3′-deoxynucleotide sequence corresponding to sequence from the 5′ end of said target RNA; and 
 quantitatively determining said target RNA by performing polymerase chain reaction (PCR) on said RNA:cDNA duplex using said first and second primers. 
 
     
     
       2. The method of  claim 1  wherein the 5′ adaptor or the tag contains deoxynucleotide “barcode” sequence to track sample identity. 
     
     
       3. The method of  claim 2  wherein the deoxynucleotide “barcode” sequence is between 2 to 15 nucleotides in length. 
     
     
       4. The method of  claim 1  wherein the quantitative determination of RNA is based on a next generation sequencing platform. 
     
     
       5. The method of  claim 1  wherein the target RNA is microRNA.